A primary limitation of bioprosthetic implants made from animal tissues is the occurrence of hyperacute rejection reactions in transplant recipients. Such reactions are driven largely by the presence of antigenic carbohydrate epitopes within implanted tissues, the most common of which is the α-GAL glycoprotein epitope: D Galactose (α 1-3) Galactose (β1-4) N acetyl Glucosamine-R-motif (α-GAL) is found on vascular endothelial tissues of all species with the exception of old world monkeys, great apes, and humans. The presence of α-GAL on harvested animal donor tissues elicits an immediate and powerful immune response after transplantation into humans that can quickly destroy surrounding tissues and/or organs. The rapidity of the rejection response is due to very high levels of preformed anti-α-GAL antibodies in human subjects (nearly 1% of all antibodies in human blood are anti-α-GAL antibodies). The high levels of anti-α-GAL are an adaptive response to the ubiquitous presence of bacteria bearing α-GAL epitopes in the human digestive tract.
The most common tissue sources for xenographic bioprosthetic tissues are equine (horse), ovine (sheep), porcine (pig) and bovine (cow) tissues, all of which bear α-GAL epitopes and are potentially antigenic. One approach for limiting the antigenicity of bioprosthetic tissues is to chemically modify antigenic epitopes so that they are no longer recognized by host antibodies. This is typically accomplished by chemical fixation, which involves exposing a bioprosthetic tissue to a fixative agent (or tanning agent) that forms cross-linkages within (intramolecular cross-linkages) and/or between (intermolecular cross-linkages) polypeptides of the tissue. Examples of fixative agents used for treating bioprosthetic tissues include formaldehyde, glutaraldehyde, dialdehyde starch, hexamethylene diisocyanate and polyepoxy compounds. Glutaraldehyde is the most widely used fixative agent and glutaraldehyde treatment is currently the standard approach for stabilizing clinically useful bioprosthetic tissues. Examples of glutaraldehyde fixed bioprosthetic heart valves include the Carpentier-Edwards® Stented Porcine Bioprosthesis, the Carpentier-Edwards® PERIMOUNT® Pericardial Bioprosthesis, and the Edwards PRIMA Plus™ Stentless Aortic Bioprosthesis, all available from Edwards Lifesciences, Irvine, Calif. 92614.
Although chemical fixation can considerably limit the antigenicity of bioprosthetic tissues, fixed tissues, particularly glutaraldehyde-fixed tissue, suffer from several drawbacks. For example, the protective effects of glutaraldehyde fixation tend to deteriorate over the lifespan of bioprosthetic implants due to the labile Schiff Base cross-links, resulting in increased immunogenicity and impaired long-term stability and performance. In addition, glutaraldehyde treatment renders bioprosthetic tissues more susceptible to calcification, particularly when an implant remains in place for an extended period of time (e.g., more than ten years) due to their high levels of residual aldehyde groups. Structural valve deterioration (SVD) is the most common cause for early valve explanation, with tissue calcification the leading cause of failure in bioprosthetic implants. These glutaraldehyde-derived aldehydes are associated with high levels of calcium mineralization.
U.S. Pat. No. 6,861,211 to Levy and Vyavahare describes methods of stabilizing a bioprosthetic tissue through chemical cross-linking affected by treating the tissue with an agent, such as periodate, that oxidizes carbohydrate moieties of glycosaminoglycans (GAG) to generate aldehydes, and then treating the tissue with a bifunctional agent that reacts with the carbohydrate aldehydes as well as reactive groups on adjacent proteins to cross-link the GAG to the surrounding tissue matrix. Like conventional glutaraldehyde fixation, the methods result in residual reactive aldehyde groups, which serve as potential calcium binding sites and thus destabilize the tissue by ultimately compromising the biomechanical properties of the material.
U.S. Pat. No. 6,383,732 (Stone) describes an alternative to chemical modification for limiting the antigenicity of bioprosthetic tissues using the enzyme alpha-galactosiadase to destroy α-GAL epitopes. Enzymatic approaches suffer from the general high cost of enzyme preparations and the fact that the large size of alpha-galactosiadase and other enzymes prevents these protein structures from penetrating deeply into tissues, such as the extracellular matrix of pericardial bioprosthetic tissues. Thus, enzyme-based treatments do not eliminate all of the epitopes targeted by an enzyme, particularly in the interior of a bioprosthetic implant. In addition, alpha-galactosiadase and other enzymes are specific for particular epitopes (e.g., α-GAL in the case of alpha galactosiadase), making it highly difficult to limit the antigenicity of tissues containing multiple and/or unknown epitopes. The enzymatic removal of cellular components and tissue structures can also degrade the biomechanical properties of the tissue. Moreover, these enzyme treatments cannot be used with glutaraldehyde-fixed tissue since the enzyme's protein structure will react with the residual aldehydes and become covalently bound to the material. The result is an increase in foreign proteins and further degradation in tissue performance.
Accordingly, there remains a need in the art for the development of new and improved methods for reducing antigenicity and limiting calcification of xenographic tissues, thereby enhancing the durability, stability, and performance of the tissues. These enhanced characteristics are consistent with the demands of bioprosthetic tissues in vivo, including maintaining the structural, mechanical, and biocompatible properties of, for example, heart valves.